Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR/anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb) interacting with the elongated FG loop of the Cbeta domain situated beneath the Vbeta domain. This long, along with partially exposed ABED beta-sheet and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. To now characterize the function of this cavity, its structural elements and associated CD3 components, four aims will be pursued. First, mutational studies in conjunction with T cell transfection will be employed to determine the role of the Cbeta FG loop, Cbeta glycans, Calpha AB loop, Cbeta Cys191 on the E strand and other "cave" residues with respect to signaling function of both class I and class II MHC-restricted TCRs. Second, the ectodomain of murine and human CD3epsilongamma and CD3epsilondelta heterodimers will be engineered from protein expression. The dimers alone or in complex with anti-CD3epsilon Fabs will be subjected to crystallization and additional structure- function analysis performed. These recombinant proteins will also be used for generation of heterodimeric specific mAbs to investigate the distribution of CD3epsilongamma and CD3epsilondelta during thymic ontogeny. Third, site-directed spin labeling of CD3epsilon and TCR Calpha or Cbeta domains will be used to investigate the movement of the TCR C module relative to the CD3epsilon component in wild type and mutant receptors following TCR V module ligation. Fourth, the role of Cbeta and pTalpha cave elements in pre-TCR function will be tested in vivo using t or mutant beta-RAG-2-/- mice as well as pTalpha-/- mice harboring variant pTalpha transgenes. Efforts will be undertaken to engineer secreted pre- TCRs for structural comparison with the TCR alphabeta heterodimer.